The long-term objectives of this proposal are to characterize genes involved in human retinal degenerations and to find possible ways to halt or eventually cure these blinding diseases. Specifically, we will study two proteins present in cones, 15A15 and retinoschisin, encoded by genes isolated previously in our laboratory, that may be interrelated in their function. We have found that the 15A15 protein binds to several DNA fragments containing the core consensus sequences for hormone response elements and that it has several features characteristic of coactivators/ co-repressors of nuclear hormone receptors (NHRs). In our first Specific Aim we will: corroborate that 15A15 plays this role studying its protein-protein interactions with GST pull-down assays;investigate if 15A15 interactions with NHRs are hormone-dependent and enhance the transactivation of endogenous NHRs;map the region of 15A15 necessary for these interactions;determine if 15A15 binds directly to response elements present in the promoter of retinoschisin and other genes expressed in cones, regulating their transcription, and if mutations in 15A15 modules abolish transcriptional enhancement of NHRs. Chromatin immunoprecipitation assays will show if 15A15 exists as part of a complex with other factors, in vivo. We will also screen the DNA of patients with retinal degenerations affecting cones for mutations in 15A15 that may result in disease. In our second Specific Aim, we will continue studying retinoschisin, the secreted protein involved in cell-cell interactions that when mutated causes X-linked juvenile retinoschisis (XLRS). We have found that cGMP, Ca2+ and possibly G-proteins may participate in regulation of secretion of retinoschisin from the photoreceptor inner segments. On the basis of these data, we will carry out a systematic study on the mechanisms that control this process, investigating the involvement of membrane and soluble guanylate cyclases (GC), the effects of GC-activating proteins, nitric oxide and carbon monoxide;the participation of rod, cone and Ca2+/calmodulin-dependent PDEs, and the role of guanine deaminase, which converts cGMP into cXMP. We will also determine whether L-type voltage gated Ca2+ channels and intracellular Ca2+ stores, as well as Gbeta-gamma and mGluR8 influence the secretion of retinoschisin. For each photoreceptor component studied, we will start with a pharmacological approach to establish its involvement, followed by its removal from the system with the use of shRNAs to corroborate its effect. Overall, our research will increase the understanding of the interaction and function of 15A15 and retinoschisin, two important proteins in cones. By learning about the biochemical pathways involved in the regulation of retinoschisin secretion in normal physiological conditions, that may be important for potential pharmacological treatment of XLRS, we hope to open new avenues to explore possible ways to rescue vision for diseases for which there are no current cures.